The components of the TCA cycle are small and very polar organic carboxylic acids. Traditional methods of reversed phase chromatography do not always yield enough retention or selectivity to confidently measure these analytes. Citric acid and isocitric acid, for example, are isobaric at 191 m/z and although malic acid has an m/z of 133, in-source fragmentation followed by decomposition in the collision cell gives rise to the same MRM transition as fumaric acid.³ To address the separation and retention of the critical pairs and very polar species of the TCA metabolites and other biologically relevant compounds, a mixed-mode chromatography method was developed. Here the ACQUITY UPLC CSH Phenyl-Hexyl 2.1 x 100 mm Column (p/n 186005407) was employed to separate lactic acid, malic acid, succinic acid, isocitric acid, citric acid, fumaric acid, pyruvic acid, α-ketoglutaric acid, phosphoenolpyruvic acid, and cis-aconitic acid, using a simple mobile phase of water and acetonitrile, each with 0.1% formic acid. Figure 1 shows the separation of the analytes at 80 °C with a flow rate of 0.4 mL/min and gradient of 2% B to 15% B over three minutes with detection performed using the Xevo TQ-S micro tandem mass spectrometer in negative ionization mode. Figure 2 shows the separation of the critical pairs: citric acid and isocitric acid, as well as malic acid and fumaric acid. Method reproducibility over different column lots was determined using a Method Validation Kit (p/n 186005580). Retention time reproducibility was less than 1.3% RSD for all of the analytes tested (Table 1). Finally, the method was tested on human urine samples by dilution with water 10 x. A sample of 3 µL was injected onto the column with the TargetLynx XS results shown in Figure 3. Here we observe well resolved components of the TCA cycle and other polar metabolites with acceptable peak shape in under three minutes.
